The present invention relates generally to the fields of chemistry and biology. More particularly, the present invention is directed to compositions and methods for use in the synthesis of oligonucleotides (e.g., DNA and RNA sequences).
A variety of synthetic approaches have been developed for preparation of oligonucleotide sequences. Typically, oligonucleotides are synthesized utilizing a building block approach which involves the sequential addition of nucleotides onto a growing oligonucleotide chain immobilized on to a solid support. Because every DNA oligonucleotide may have any of 4 different initial nucleotides, it is necessary to maintain a supply of 4 different nucleoside (A, C, G and T) loaded solid supports to be able to synthesize any given DNA sequence. In the case of DNA synthesis, the first nucleoside from the 3' end of the DNA sequence is typically preloaded on the solid support through an ester linkage. For example, if the sequence that is to be synthesized contains a T nucleoside at the 3' end, a T support is employed and the balance of the nucleotides in the DNA sequence added thereto (for example, using an automated DNA synthesizer). At the end of the total DNA synthesis, the oligonucleotide is cleaved from the solid support through the hydrolysis of the ester linkage. Taking into consideration RNA synthesis procedures, an additional 4-different nucleoside loaded solid supports must be available to the user. Similar considerations apply if any specialty modified nucleoside is desired at the 3' end.
Maintaining a supply of at least 8 different prederivatized solid supports is inconvenient and expensive. An additional consideration is the relatively short shelf life of nucleoside derivatized solid supports. Typically, after one year storage such solid supports are not longer usable. There is also the possibility that synthetic procedures may be initiated mistakenly with the wrong support leading to disastrous consequences in the final applications of the oligonucleotides.
In order to alleviate these problems some researchers have pursued developing some type of universal solid support. For example, deBear et al. derivatized glass supports with 2' (3')-O-benzoyluridine 5'-O-succinyl so that the uridine moiety is linked to the glass via an ester (succinate) linkage. de Bear et al., Nucleosides and Nucleotides 6, 821-830 (1987)!. Oligonucleotide synthesis takes place by adding nucleotide monomers to the 2' or 3' position of the uridine. Following the synthesis, the new oligonucleotides can be released from the glass, deprotected and cleaved from the uridylyl terminus in one reaction. Since the uridyl functionality is cleaved from the solid support in this cleaving reaction, the support is not available for subsequent oligonucleotide syntheses.
Crea and Horn suggested a similar approach which involved preparing the dimer 5'-O-p-chlorophenylphospho-2' (3')-O-acetyluridilyl-2' (3')-3'!-5'-O-dimethoxytritylthymidine p-chlorophenylester and attaching the dimer to cellulose via a phosphate linkage. The 5' position of the thymidine is available for oligonucleotide attachment and synthesis. R. Crea & T. Horn, Nucleic Acids Research 8, 2331 (1980)!. The subsequent use of aqueous concentrated ammonia results in the release of the synthesized oligonucleotide from the cellulose leaving the uridine portion of the dimer attached to the cellulose. Although Crea and Horn utilized the reactive vicinal groups on the uridine as the release site for the oligonucleotide from the uridine the solid support suggested in this reference is not a universal solid support since the initial oligonucleotide is incorporated in the solid support reagent and a different support is required for oligonucleotides incorporating different first nucleoside.
More recently, Schwartz et al. attached an adapter, 2' (3')-O-dimethoxytrityl-3' (2')-O-benzoyluridine-5'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite, to a thymidine derivatized polystyrene and synthesized an oligonucleotide from the O-dimethoxytrityl position of the uridine. M. E. Schwartz, R. R. Breaker, G. T. Asteriadis, and G. R. Gough, Tetrahedron Letters, Vol. 36, No. 1, pp 27-30, 1995! While this approach provides a universal solid support for oligonucleotide synthesis, the cleaving step releases the adapter and the thymidine from the support and then cleaves the synthesized oligonucleotide from the uridine. Thus, the purification process requires removing the thymidine linker and the cleaving processes leaves the solid support unavailable for subsequent uses.
The aforementioned solid supports and methods for their use disadvantages in terms of the convenience and efficiency of the subsequent oligonucleotide cleaving steps. When ammonia which has been widely accepted as a safe reagent for DNA synthesis is utilized for cleaving, as taught by deBear et al., the cleavage time is as long as 24 hours at 65.degree. C. In view of the growing trend to produce oligonucleotides as quickly as possible, this is an unacceptably long period of time. Decreasing the time required for cleaving the uridylyl from an oligonucleotide at the uridine 3' position typically uses a lead (II) ion catalyst system or the action of strong alkali hydroxides. Necessarily these processes require a separate isolation step to remove the lead ion. Additionally, when strong alkali bases are used in the cleaving processes, considerable side reactions in the form of cytosine deamination occur.
Recent improvements in the automation of simultaneous multiple oligonucleotide synthesis (whereby several oligonucleotides are synthesized simultaneously) further highlight the desirabilty of using a single universal solid support for the synthesis of oligonucleotides. Any requirement to dispense several different nucleoside preloaded solid supports on a DNA synthesizer clearly complicates the synthesis automation. Therefore, it would be highly desirable to have a universal solid support to synthesize any nucleic acid.
It is an object of the present invention to provide methods and compositions for use in the synthesis of oligonucleotides which ameliorate some of the problems encountered with the prior art methods and compositions. More particularly, it is an objective of the present invention to provide reusable oligonucleotide synthesis reagents which can be used for synthesizing a variety of oligonucleotide independent of the initial nucleoside. It is another objective to provide cleaving reagents and cleaving methods which substantially decrease the time required to cleave oligonucleotides after synthesis.